Method of improving the properties of a flour dough, a flour dough improving composition and improved food products

ABSTRACT

A method of improving the rheological properties of a flour dough and the quality of the finished product made from such a dough, comprising adding an effective amount of an oxidoreductase capable of oxidizing maltose, in particular a hexose oxidase, e.g. isolated from an algal species such as  Iridophycus flaccidum, Chondrus crispus  or  Euthora cristata  and a dough improving composition comprising the oxidoreductase.

CROSS-REFERENCE TO RELATED APPLICATIONS:

This is a divisional of application Ser. No. 09/932,923, filed Aug. 21,2001, now U.S. Pat. No. 6,726,942, which was a continuation ofapplication Ser. No. 08/676,186, filed Sep. 12, 1996, now U.S. Pat. No.6,358,543, which was a continuation-in-part of application Ser. No.08/483,870, filed Jun. 7, 1995, abandoned, and was a U.S. national phaseof PCT/DK96/00239, filed Jun. 4, 1996.

FIELD OF THE INVENTION

The invention pertains to the provision of flour doughs having improvedrheological properties and farinaceous food products having improvedquality characteristics and it provides a maltose oxidizingoxidoreductase-containing composition capable of conferring suchimproved properties on doughs and finished food products made herefromwhen it is added as a component to the doughs, and a method of preparingimproved doughs and farinaceous food products.

TECHNICAL BACKGROUND AND PRIOR ART

The invention relates in particular to a method of providing flourdoughs having improved rheological properties and to finished baked ordried products made from such doughs, which have improved textural,eating quality and dimensional characteristics.

In this connection, the “strength” or “weakness” of doughs is animportant aspect of making farinaceous finished products from doughs,including baking. The “strength” or “weakness” of a dough is primarilydetermined by its content of protein and in particular the content andthe quality of the gluten protein is an important factor in thatrespect. Flours with a low protein content is generally characterized as“weak”. Thus, the cohesive, extensible, rubbery mass which is formed bymixing water and weak flour will usually be highly extensible whensubjected to stress, but it will not return to its original dimensionswhen the stress is removed.

Flours with a high protein consent are generally characterized as“strong” flours and the mass formed by mixing such a flour and waterwill be less extensible than the mass formed from a weak flour, andstress which is applied during mixing will be restored without breakdownto a greater extent than is the case with a dough mass formed from aweak flour. Strong flour is generally preferred in most baking contextsbecause of the superior rheological and handling properties of the doughand the superior form and texture qualities or the finished baked ordried products made from the strong flour dough.

Doughs made from strong flours are generally more stable. Stability of adough is one of the most important characteristics of flour doughs.According to American Association of Cereal Chemists (AACC) Method36-01A the term “stability” can be defined as “the range of dough timeover which a positive response is obtained and that property of arounded dough by which it resists flattening under its own weight over acourse of time”. According to the same method, the term “response” isdefined as “the reaction of dough to a known and specific stimulus,substance or set of conditions, usually determined by baking it incomparison with a control”

Within the bakery and milling industries it is known to use dough“conditioners” to strengthen the dough. Such dough conditioners arenormally non-specific oxidizing agents such as eg iodates, peroxides,ascorbic acid, K-bromate or azodi-carbonamide and they are added todough with the aims of improving the baking performance of flour toachieve a dough with improved stretchability and thus having a desirablestrength and stability. The mechanism behind this effect of oxidizingagents is that the flour proteins, in particular gluten contains thiolgroups which, when they become oxidized, form disulphide bonds wherebythe protein forms a more stable matrix resulting in a better doughquality and improvements of the volume and crumb structure of the bakedproducts.

In addition to the above usefulness of ascorbic acid/ascorbate as acough conditioner due to its oxidizing capacity, these compounds mayalso act as substrate for an oxidoreductase, ascorbate oxidase which isdisclosed in EP 0 582 116 A1. In the presence of its substrate, thisenzyme converts ascorbic acid/ascorbate to dehydroascorbic acid andH₂O₂. This prior art does not suggest that ascorbic acid oxidase in thepresence of ascorbic acid/ascorbate might have a dough conditioningeffect, but assumingly this is the case.

However, the use of several of the currently available oxidizing agentsis either objected to by consumers or is not permitted by regulatorybodies and accordingly, it has been attempted to find alternatives tothese conventional flour and dough additives and the prior art has i.a.suggested the use of glucose oxidase for this purpose.

Thus, U.S. Pat. No. 2,783,150 discloses the addition of glucose oxidaseto flour to improve dough strength and texture and appearance of bakedbread.

CA 2,012,723 discloses bread improving compositions comprisingcellulolytic enzymes such as xylanases and glucose oxidase, the latterenzyme being added to reduce certain disadvantageous effects of thecellulolytic enzymes (reduced dough strength and stickiness) and it isdisclosed that addition of glucose to the dough is required to obtain asufficient glucose oxidase activity.

JP-A-92-084848 suggests the use of a bread improving compositioncomprising glucose oxidase and lipase.

EP-B1-321 811 discloses the use of an enzyme composition comprisingsulfhydryl oxidase and glucose oxidase to improve the rheologicalcharacteristics of doughs. It is mentioned in this prior art documentthat the use of glucose oxidase alone has not been successful.

In EP-B1-338 452 is disclosed an enzyme composition for improving doughstability, comprising a mixture of cellulase/hemicellulase, glucoseoxidase and optionally sulfhydryl oxidase.

However, the use of glucose oxidase as a dough improving additive hasthe limitation that this enzyme requires the presence of sufficientamounts of glucose as substrate in order to be effective in a doughsystem and generally, the glucose content in cereal flours is low.Therefore, the absence of glucose in doughs or the low content hereof indoughs will be a limiting factor for the effectiveness of glucoseoxidase as a dough improving agent.

In contrast hereto, the content of maltose in cereal flours is generallysignificantly higher than that of glucose and accordingly, a freshlyprepared dough will normally contain more maltose than glucose. Thus, inan experiment where the content of sugars in supernatants fromsuspensions of wheat flour and a dough prepared from the flour andfurther comprising water, yeast, salt and sucrose (as described in thefollowing example 2.3) were analyzed, the following values (% by weightcalculated on flour) were found:

Flour Dough Sucrose 0.3 <0.01 Galactose 0.001 0.01 Glucose 0.25 0.72Maltose 2.6 1.4 Fructose 0.08 0.67 Lactose <0.01 <0.01

In addition, the content of maltose remains at a relatively high levelin a dough which is leavened by yeast, since the yeast primarilyutilizes glucose, or it may even increase in the dough e.g. duringproofing due to the activity of starch degrading enzymes such as e.g.β-amylase, which is inherently present in the flour or is added to thedough.

Whereas the prior art has recognized the useful improving effects ofglucose oxidase on the rheological characteristics of bread doughs andon the quality of the corresponding baked products, it has also beenrealized that the use of this enzyme has several drawbacks. Thus, it maybe required to add sucrose or glucose as substrate to the dough toobtain a sufficient effect and glucose oxidase does not constantlyprovide a desired dough or bread improving effect when used alonewithout the addition of other enzymes.

However, it has now been found that the addition of an oxidoreductase,which is capable of oxidizing maltose, including hexose oxidase as asole dough conditioning agent, i.e. without concomitant addition ofsubstrate for the added enzyme, or of other enzymes, to a farinaceousdough results in an increased resistance hereof to breaking when thedough is stretched, i.e. this enzyme confers in itself to the dough anincreased strength whereby the dough becomes less prone to mechanicaldeformation. It is contemplated that this effect of addition of hexoseoxidase to a dough is the result of the formation of cross-links betweenthiol groups in sulphur-containing amino acids in wheat gluten whichoccurs when the H₂O₂ generated by the enzyme in the dough reacts withthe thiol groups which are hereby oxidized.

Hexose oxidase (D-hexose:O₂-oxidoreductase, EC 1.1.3.5) is an enzymewhich in the presence of oxygen is capable of oxidizing D-glucose andseveral other reducing sugars including maltose, glucose, lactose,galactose, xylose, arabinose and cellobiose to their correspondinglactones with subsequent hydrolysis to the respective aldobionic acids.Accordingly, hexose oxidases differ from glucose oxidase which can onlyconvert D-glucose, in that hexose oxidases can utilize a broader rangeof sugar substrates. The oxidation catalyzed by the enzyme can beillustrated as follows:D-Glucose+O₂→δ-D-gluconolactone+H₂O₂, orD-Galactose+O₂→γ-D-galactogalactone+H₂O₂

Hexose oxidase (in the following also referred to as HOX) has beenisolated from several red algal species such as Iridophycus flaccidum(Bean and Hassid, 1956, J. Biol. Chem., 218:425-436) and Chondruscrispus (Ikawa 1982, Methods Enzymol., 89:145-149). Additionally, thealgal species Euthora cristata (Sullivan et al. 1973, Biochemica etBiophysica Acta, 309:11-22) has been shown to produce HOX.

Other potential sources of hexose oxidase according to the inventioninclude microbial species or land-growing plant species. Thus, as anexample of such a plant source, Bean et al., Journal of BiologicalChemistry (1961) 236: 1235-1240, have disclosed an oxidoreductase fromcitrus fruits which is capable of oxidizing a broad range of sugarsincluding D-glucose, D-galactose, cellobiose, lactose, maltose,D-2-deoxyglucose, D-mannose, D-glucosamine and D-xylose. Another exampleof an enzyme having hexose oxidase activity is the enzyme system ofMalleomyces mallei disclosed by Dowling et al., Journal of Bacteriology(1956) 72:555-560.

It has been reported that hexose oxidase isolated from the above naturalsources may be of potential use in the manufacturing of certain foodproducts. Thus, hexose oxidase isolated from Iridophycus flaccidum hasbeen shown to be capable of converting lactose in milk with theproduction of the corresponding aldobionic acid and has been shown to beof potential interest as an acidifying agent in milk, e.g. to replaceacidifying microbial cultures for that purpose (Rand, 1972, Journal ofFood Science, 37:698-701). In that respect, hexose oxidase has beenmentioned as a more interesting enzyme than glucose oxidase, since thislatter enzyme can only be enzymatically effective in milk or other foodproducts not containing glucose or having a low content of glucose, ifglucose or the lactose-degrading enzyme lactase which convert thelactose to glucose and galactose, is also added.

The capability of oxidoreductases including that of hexose oxidase togenerate hydrogen peroxide has also been utilized to improve the storagestability of certain food products including cheese, butter and fruitjuice as it is disclosed in-JP-B-73/016612. It has also been suggestedthat oxidoreductases may be potentially useful as antioxidants in foodproducts.

However, the present invention has demonstrated that hexose oxidase ishighly useful as a dough conditioning agent in the manufacturing offlour dough products including not only bread products but also otherproducts made from flour doughs such as noodles and alimentary pasteproducts.

SUMMARY OF THE INVENTION

Accordingly, the invention relates in a first aspect to a method ofimproving the rheological properties of a flour dough and the quality ofthe finished product made from the dough, comprising adding to the doughingredients, dough additives or the dough an effective amount of anoxidoreductase which at least is capable of oxidizing maltose, such ase.g. a hexose oxidase.

In a further aspect, there is also provided a dough bakery productimproving composition comprising an oxidoreductase which at least iscapable of oxidizing maltose, and at least one further dough ingredientor dough additive.

In still further aspects, the invention pertains to a method ofpreparing a bakery product, comprising preparing a flour dough includingadding an effective amount of an oxidoreductase which at least iscapable of oxidizing maltose and baking the dough, and a method ofpreparing a dough-based food product comprising adding to the dough aneffective amount of a maltose oxidizing oxidoreductase.

DETAILED DISCLOSURE OF THE INVENTION

In one aspect, the present method contemplates a method of improving therheological properties of flour doughs. The method comprises, as it ismentioned above, the addition of an effective amount of a maltoseoxidizing oxidoreductase either to a component of the dough recipe or tothe dough resulting from mixing all of the components for the dough. Inthe present context, “an effective amount” is used to indicate that theamount is sufficient to confer to the dough and/or the finished productimproved characteristics as defined herein.

In one useful embodiment of the method according to the invention, theoxidoreductase is a hexose oxidase. Hexose oxidase can, as it isdescribed in details herein, be isolated from marine algal speciesnaturally producing that enzyme. Such species are found in the familyGigartinaceae which belong to the order Gigartinales. Examples of hexoseoxidase producing algal species belonging to Gigartinaceae are Chondruscrispus and Iridophycus flaccidum. Also algal species of the orderCryptomeniales including the species Euthora cristata are potentialsources of hexose oxidase.

When using such natural sources for hexose oxidase, the enzyme istypically isolated from the algal starting material by extraction usingan aqueous extraction radium. As starting material may be used algae intheir fresh state as harvested from the marine area where they grow, orthe algal material can be used for extraction of hexose oxidase afterdrying the fronds e.g. by air-drying at ambient temperatures or by anyappropriate industrial drying method such as drying in circulated heatedair or by freeze-drying. In order to facilitate the subsequentextraction step, the fresh or dried starting material may advantageouslybe comminuted e.g. by grinding or blending.

As the aqueous extraction medium, buffer solutions e.g. having a pH inthe range of 5-8, such as 0.1 M sodium phosphate buffer, 20 mMtriethanolamine buffer or 20 mM Tris-HCl buffer are suitable. The hexoseoxidase is typically extracted from the algal material by suspending thestarting material in the buffer and keeping the suspension at atemperature in the range of 0-20° C. such as at about 50° C. for 1 to 5days, preferably under agitation.

The suspended algal material is then separated from the aqueous mediumby an appropriate separation method such as filtration, sieving orcentrifugation and the hexose oxidase is subsequently recovered from thefiltrate or supernatant. Optionally, the separated algal material issubjected to one or more further extraction steps.

Since several marine algae contain coloured pigments such asphycocyanins, it may be required to subject the filtrate or supernatantto a further purification step whereby these pigments are removed. As anexample, the pigments may be removed by treating the filtrate orsupernatant with an organic solvent in which the pigments are solubleand subsequently separating the solvent containing the dissolvedpigments from the aqueous medium. Alternatively, pigments may be removedby subjecting the filtrate or supernatant to a hydrophobic interactionchromatography step.

The recovery of hexose oxidase from the aqueous extraction medium iscarried out by any suitable conventional methods allowing isolation ofproteins from aqueous media. Such methods, examples of which will bedescribed in details in the following, include conventional methods forisolation of proteins such as ion exchange chromatography, optionallyfollowed by a concentration step such as ultrafiltration. It is alsopossible to recover the enzyme by adding substances such as e.g.(NH₄)₂SO₄ or polyethylene glycol (PEG) which causes the protein toprecipitate, followed by separating the precipitate and optionallysubjecting it to conditions allowing the protein to dissolve.

For certain applications of hexose oxidase it is desirable to providethe enzyme in a substantially pure form e.g. as a preparationessentially without other proteins or non-protein contaminants andaccordingly, the relatively crude enzyme preparation resulting from theabove extraction and isolation steps may be subjected to furtherpurification steps such as further chromatography steps, gel filtrationor chromato-focusing as it will also be described by way of example inthe following.

In a preferred embodiment of the method according to the invention, aflour dough is prepared by mixing flour with water, a leavening agentsuch as yeast or a conventional chemical leavening agent, and aneffective amount of hexose oxidase under dough forming conditions. Itis, however, within the scope of the invention that further componentscan be added to the dough mixture.

Typically, such further dough components include conventionally useddough components such as salt, a sweetening agent such as sugars, syrupsor artificial sweetening agents, lipid substances including shortening,margarine, butter or an animal or vegetable oil and one or more doughadditives such as emulsifying agents, starch degrading enzymes,cellulose or hemicellulose degrading enzymes, proteases, lipases,non-specific oxidizing agents such as those mentioned above, flavouringagents, lactic acid bacterial cultures, vitamins, minerals,hydrocolloids such as alginates, carrageenans, pectins, vegetable gumsincluding e.g. guar gum and locust bean gum, and dietary fibersubstances.

Conventional emulsifiers used in making flour dough products include asexamples monoglycerides, diacetyl tartaric acid esters of mono- anddiglycerides of fatty acids, and lecithins e.g. obtained from soya.Among starch degrading enzymes, amylases are particularly useful asdough improving is additives. α-amylase breaks down starch into dextrinswhich are further broken down by β-amylase into maltose. Other usefulstarch degrading enzymes which may be added to a dough compositioninclude glucoamylases and pullulanases. In the present context, furtherinteresting enzymes are xylanases and other oxidoreductases such asglucose oxidase, pyranose oxidase and sulfhydryl oxidase.

A preferred flour is wheat flour, but doughs comprising flour derivedfrom other cereal species such as from rice, maize, barley, rye anddurra are also contemplated.

The dough is prepared by admixing flour, water, the oxidoreductaseaccording to the invention and other possible ingredients and additives.The oxidoreductase can be added together with any dough ingredientincluding the water or dough ingredient mixture or with any additive oradditive mixture. The dough can be prepared by any conventional doughpreparation method common in the baking industry or in any otherindustry making flour dough based products.

The oxidoreductase can be added as a liquid preparation or in the formor a dry powder composition either comprising the enzyme as the soleactive component or in admixture with one or more other doughingredients or additive. The amount of the enzyme component addednormally is an amount which results in the presence in the finisheddough of 1 to 10,000 units per kg of flour, preferably 5 to 5000 unitssuch as 10 to 1000 units. In useful embodiments, the amount is in therange of 20 to 500 units per kg of flour. In the present context 1oxidoreductase unit corresponds to the amount of enzyme which underspecified conditions results in the conversion of 1 μmole glucose perminute. The activity is stated as units per g of enzyme preparation.

The effect of the oxidoreductase on the rheological properties of thedough can be measured by standard methods according to the InternationalAssociation of Cereal Chemistry (ICC) and the American Association ofCereal Chemistry (AACC) including the amylograph method (ICC 126), thefarinograph method (AACC 54-21) and the extensigraph method (AACC54-10). The extensigraph method measures e.g. the doughs ability toretain gas evolved by yeast and the ability to withstand proofing. Ineffect, the extensigraph method measures the relative strength of adough. A strong dough exhibits a higher and, in some cases, a longerextensigraph curve than does a weak dough. AACC method 54-10 defines theextensigraph in the following manner: “the extensigraph records aload-extension curve for a test piece of dough until it breaks.Characteristics of load-extension curves or extensigrams are used toassess general quality of flour and its responses to improving agents”.

In a preferred embodiment or she method according to the invention, theresistance to extension of the dough in terms of the ratio between theresistance to extension (height of curve, B) and the extensibility(length of curve, C) , i.e. the B/C ratio as measured by the AACC method54-10 is increased by at least 10% relative to that of an otherwisesimilar dough not containing oxidoreductase. In more preferredembodiments, the resistance to extension is increased by at least 20%,such as at least 50% and in particular by at least 100%.

The method according to the invention can be use for any type of flourdough with the aims of improving the rheological properties hereof andthe quality of the finished products made from the particular type ofdough. Thus, the method is highly suitable for the making ofconventional types of yeast leavened bread products including wheatflour based bread products such as loaves and rolls. However, it iscontemplated that the method also can improve the properties of doughsin which leavening is caused by the addition of chemical leaveningagents, including sweet bakery products such as cake products includingas examples pound cakes and muffins, or scones.

In one interesting aspect, the invention is used to improve therheological properties of doughs intended for noodle products including“white noodles” and “chinese noodles” and to improve the texturalqualities of the finished noodle products. A typical basic recipe forthe manufacturing of noodles comprises the following ingredients: wheatflour 100 parts, salt 0.5 parts and water 33 parts. The noodles aretypically prepared by mixing the ingredients in an appropriate mixingapparatus followed by rolling out the noodle dough using an appropriatenoodle machine to form the noodle strings which are subsequently airdried.

The quality of the finished noodles is assessed ia by their colour,cooking quality and texture. The noodles should cook as quickly aspossible, remain firm after cooking and should preferably not loose anysolids to the cooking water. On serving the noodles should preferablyhave a smooth and firm surface not showing stickiness and provide a firm“bite” and a good mouthfeel. Furthermore, it is important that thenoodles have a light colour.

Since the appropriateness of wheat flour for providing noodles havingthe desired textural and eating qualities may vary according to the yearand the growth area, it is usual to add noodle improvers to the dough inorder to compensate for sub-optimal quality of the flour. Typically,such improvers will comprise dietary fiber substances, vegetableproteins, emulsifiers and hydrocolloids such as e.g. alginates,carrageenans, pectins, vegetable gums including guar gum and locust beangum, and amylases.

It has been attempted to use glucose oxidase as a noodle improvingagent. However, as mentioned above, the content of glucose may be so lowin wheat flour that this enzyme will not be effective.

It is therefore an important aspect of the invention that theoxidoreductase according to the invention is useful as a noodleimproving agent, optionally in combination with other componentscurrently used to improve the quality of noodles. Thus, it iscontemplated that noodles prepared in accordance with the above methodwill have improved properties with respect to colour, cooking and eatingqualities including a firm, elastic and non-sticky texture andconsistency.

In a further useful embodiment the dough which is prepared by the methodaccording to the invention is a dough for preparing an alimentary pasteproduct. Such products which include as examples spaghetti and maccaroniare typically prepared from a dough comprising as the main ingredientsflour and eggs. After mixing of the ingredient, the dough is formed tothe desired type of paste product and air dried. It is contemplated thatthe addition to a paste dough will have a significant improving effecton the extensibility and stability hereof resulting in finished pasteproduct having improved textural and eating qualities.

In a further aspect of the invention there is provided a dough improvingcomposition comprising the oxidoreductase according to the invention andat least one further dough ingredient or dough additive.

In a preferred embodiment, the oxidoreductase is hexose oxidase. Thefurther ingredient or additive can be any of the ingredients oradditives which are described above. The composition may conveniently bea liquid preparation comprising the oxidoreductase. However, thecomposition is conveniently in the form of dry composition. It will beunderstood that the amount of oxidoreductase activity in the compositionwill depend on the types and amounts of the further ingredients oradditives. However, the amount of oxidoreductase activity is preferablyin the range of 10 to 100,000 units, preferably in the range of 100 to50,000 units such as 1,000 to 10,000 units including 2,000 to 5,000units.

Optionally, the composition may be in the form of a complete doughadditive mixture or pre-mixture for a making a particular finishedproduct and containing all of the dry ingredients and additives for sucha dough. In specific embodiments, the composition may be oneparticularly useful for preparing a baking product or in the making of anoodle product or an alimentary paste product.

As mentioned above, the present invention provides a method forpreparing a bakery product including the addition to the dough of anoxidoreductase such as e.g. hexose oxidase. In particular, this methodresults in bakery products such as the above mentioned products in whichthe specific volume is increased relative to an otherwise similar bakeryproduct, prepared from a dough not containing oxidoreductase. In thiscontext, the expression “specific volume” is used to indicate the ratiobetween volume and weight of the product. It has surprisingly been foundthat in accordance with the above method, the specific volume can beincreased significantly such as by at least 10%, preferably by at least20%, including by at least 30%, preferably by at least 40% and morepreferably by at least 50%.

In one advantageous embodiment of the above method at least one furtherenzyme is added to the dough. Suitable examples hereof include acellulose, a hemicellulase, a xylanase, a starch degrading enzyme, aglucose oxidase, a lipase and a protease.

The invention will now be described by way of illustration in thefollowing non-limiting examples and the drawing in which

FIG. 1 illustrates the changes of the B/C ratio relative to controldough without enzyme in a dough to which was added 100 units/kg flour ofhexose oxidase (black columns) or 100 units/kg flour of glucose oxidase(grey columns) after 45, 90 and 135 minutes, respectively as measured bythe AACC extensigraph method 54-10.

EXAMPLE 1

1.1. Purification of Hexose Oxidase from Chondrus crispus

A purified hexose oxidase preparation was obtained using the belowextraction and purification procedures. During these procedures and thefollowing characterizations of the purified enzyme, the following assayfor determination of hexose oxidase activity was used:

1.1.1. Assay of Hexose Oxidase Activity

The assay was based on the method described by Sullivan and Ikawa(Biochimica et Biophysica Acta, 1973, 309:11-22), but modified to run inmicrotiter plates. An assay mixture contained 150 μl β-D-glucose (0.1 Min 0.1 M sodium phosphate buffer, pH 6.3), 120 μl 0.1 M sodium phosphatebuffer, pH 6.3, 10 μl o-dianisidine-dihydrochloride (Sigma D-3252, 3.0mg/ml in H₂O), 10 μl peroxidase (POD) (Sigma P-8125, 0.1 ml in 0.1 Msodium phosphate buffer, pH 6.3) and 10 μl enzyme (HOX) solution. Blankswere made by adding buffer in place of enzyme solution.

The incubation was started by the addition of glucose. After 15 minutesof incubation at 25° C. the absorbance at 405 nm was read in an ELISAreader. A standard curve was constructed using varying concentrations ofH₂O₂ in place of the enzyme solution.

The reaction can be described in the following manner:

H₂O₂+o-dianisidine_(red)→2 H₂O+o-dianisidine_(ox)

Oxidized o-dianisidine has a yellow colour absorbing at 405 nm .

1.1.2. Extraction

Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. This fresh material was homogenized in a pin mill(Alpine). To a 100 g sample of the resulting homogenized frond materialwas added 300 ml of 0.1 M sodium phosphate buffer, pH 6.8. The mixturewas subsequently sonicated in a sonication bath for 5 minutes and thenextracted under constant rotation for 4 days at 50° C., followed bycentrifugation of the mixture at 47,000×g for 20 minutes.

300 ml of the resulting clear pink supernatant was desalted byultrafiltration using an Amicon ultrafiltration unit equipped with anOmega (10 kD cut off, Filtron) ultrafiltration membrane.

1.1.3.. Anion Exchange Step

The retentate resulting from 1.1.2 was applied to a 5×10 cm column with200 ml Q-Sepharose FF equilibrated in 20 mM triethanolamine, pH 7.3. Thecolumn was washed with the equilibration buffer and hexose oxidaseeluted with a 450 ml gradient of 0 to 1 M of NaCl in equilibrationbuffer. The column was eluted at 6 ml/minute, and fractions of 14 mlcollected. Fractions 9-17 (total 125 ml) were pooled and concentrated byultrafiltration using an Amicon 8400 unit equipped with an Omega (10 kDcut off, Filtron) ultrafiltration membrane to 7.5 ml.

1.1.4. Gel Filtration

The above 7.5 ml retentate was applied to a Superdex 200 2.6×60 cm gelfiltration column equilibrated in 50 mM sodium phosphate buffer, pH 6.4and eluted at a flow rate of 1 ml/-minute. Fractions of 4 ml werecollected. Fractions 17-28 (total volume 50 ml) containing the hexoseoxidase activity were pooled.

1.1.5. Hydrophobic Interaction Chromatography

To the pool resulting from the gel filtration step 1.1.4 ammoniumsulphate was added to a final concentration of 2 M. This mixture wasthen applied to a 1.6×16 cm column with 32 ml phenyl sepharoseequilibrated in 20 mM sodium phosphate buffer, pH 6.3 and 2 M (N₄)₂SO₄.The column was washed with equilibration buffer followed by elution ofhexose oxidase at a flow rate of 2 ml/minute using a 140 linear gradientfrom 2 M to 0 M (NH₄)₂SO₄ in 20 mM sodium phosphate buffer. Fractions of4 ml were collected and fractions 24-33 containing the hexose oxidaseactivity were pooled.

The above mentioned pink colour accompanies the enzyme, but it isseparated from hexose oxidase in this purification step.

1.1.6. Mono Q Anion Exchange

The above pool resulting from the above phenyl sepharose chromatographystep was desalted by ultrafiltration as described above. 2 ml of thispool was applied to a Mono Q, HR 5/5 column equilibrated in 20 mM,triethanolamine, pH 7.3. The column was subsequently eluted using a 45ml linear gradient from 0 to 0.65 M NaCl in equilibration buffer at aflow rate 1.5 ml/minute. Fractions of 1.5 ml were collected andfractions 14-24 were pooled.

1.1.7. Mono P Anion Exchange

The hexose oxidase-containing pool from the above step 1.1.6 was appliedto a Mono P HR 5/5 column equilibrated in 20 mM bis-Tris buffer, pH 6.5.The enzyme was eluted using a 45 ml linear gradient from 0 to 0.65 MNaCl in equilibration buffer at a flow rate of 1.5 ml/minute, andfractions of 0.75 ml were collected. The highest hexose oxidase activitywas found in fraction 12.

1.2. Characterization of the Purified Hexose Oxidase

The hexose oxidase-containing pools from the above steps 1.1.6 and 1.1.7were used in the below characterization experiments:

1.2.1. Determination of Molecular Weight

The size of the purified native hexose oxidase was determined by gelpermeation chromatography using a Superose 6 HR 10/30 column at a flowrate of 0.5 ml/minute in 50 mM sodium phosphate buffer, pH 6.4. Ferritin(440 kD), catalase (232 kD), aldolase (158 kD), bovine serum albumin (67kD) and chymotrypsinogen (25 kD) were used as size standards. Themolecular weight of the purified hexose oxidase was determined to be120±10 kD.

1.2.2. Determination of pH Optimum

Assay mixtures for the determination of pH optimum (final volume 300 μl)contained 120 μl of 0.1 M stock solution of sodium phosphate/citratebuffer of varying pH values. All other assay mixture components weredissolved in H₂O. The pH was determined in the diluted stock buffersolutions at 25° C.

The hexose oxidase showed enzymatic activity from pH 3 to pH 8, but withoptimum in the range of 3.5 to 5.5.

1.2.3. K_(m) of the Hexose Oxidase for Glucose and Maltose, Respectively

Kinetic data were fitted to v=V_(max)S/(K_(m)+S) were V_(max) is themaximum velocity, S is the substrate concentration and K_(m) is theconcentration giving 50% of the maximum rate (Michaelis constant) usingthe EZ-FIT curve fitting microcomputer programme (Perrella, F. W., 1988,Analytical Biochemistry, 174:437-447).

A typical hyperbolic saturation curve was obtained for the enzymeactivity as a function of glucose and maltose, respectively. K_(m) forglucose was calculated to be 2.7 mM±0.7 mM and for maltose the K_(m) wasfound to be 43.7±5.6 mM.

EXAMPLE 2

Dough Improving Effect of Hexose Oxidase Extracted from Chondrus crispus

2.1. Purification of Hexose Oxidase from Chondrus crispus

For this experiment, hexose oxidase was prepared in the followingmanner:

Fresh Chondrus crispus material was collected at the coast of Brittany,France. The material was freeze-dried and subsequently ground. 40 g ofthis ground material was suspended in 1000 ml of 20 mM triethanolamine(TEA) buffer, pH 7.3 and left to stand at 5° C. for about 64 hours withgentle agitation and then centrifuged at 2000×g for 10 minutes. Thesupernatant was filtered through GF/A and GF/C glass filters followed byfiltering through a 45 μm pore size filter to obtain a filtratepreparation of 800 ml having hexose oxidase activity corresponding to aglucose oxidase activity of 0.44 units per g of preparation. Theactivity was determined using the below procedure.

The supernatant was applied onto a 330 ml bed volume chromatographiccolumn with anionic exchange Q Sepharose Big Beads (dead volume 120 ml).The bound proteins were eluted over 180 minutes using a gradient from 0to 0.5 M NaCl in 20 mM TEA buffer, pH 7.3 followed by 1 M NaCl in 20 mMTEA buffer, and fractions of 9 ml were collected and analyzed for hexoseoxidase activity using the below analytical procedure.

Hexose oxidase activity-containing fractions 60-83 were pooled (about250 ml) and concentrated and desalted by ultrafiltration to about 25 ml.This step was repeated twice on the retentates to which was added 100 ml0.05 mM TEA. The resulting retentate of 25 ml contained 0.95 glucoseoxidase activity units per g.

2.2. Determination of Glucose Oxidase Activity

Definition: 1 glucose oxidase (GOD) unit corresponds to the amount ofenzyme which under the specified conditions results in the conversion of1 μmole glucose per min. The activity is stated as units per g of enzymepreparation.

Reagents: (i) Buffer: 20 g Na₂HPO₄-2H₂O is dissolved in 900 ml distilledwater, pH is adjusted to 6.5; (ii) dye reagent (stock solution): 200 mgof 2,6-dichloro-phenol-indophenol, Sigma No. D-1878 is dissolved in 1000ml distilled water under vigorous agitation for 1 hour; (iii) peroxidase(stock solution): Boehringer Mannheim No. 127 361, 10,000 units isdissolved in 10 ml distilled water and 4.2 g of ammonium sulphate added;(iv) substrate: 10% w/v D-glucose solution in buffer, (v) standardenzyme: hydrase #1423 from Amano.

Analytical principle and procedure: Glucose is converted to gluconicacid and H₂O₂ which is subsequently converted by peroxidase to H₂O andO₂. The generated oxygen oxidizes the blue dye reagent2,6-dichloro-phenol-indophenol which thereby changes its colour topurple. The oxidized colour is measured spectrophotometrically at 590 nmand the enzymatic activity values calculated relative to a standard.

2.3. The Effect of the Hexose Oxidase Preparation on Cross-LinkingBetween Thiol Groups in a Wheat Flour Based Dough

The effect of hexose oxidase on the formation of thiol groupcross-linking was studied by measuring the content of free thiol groupsin a dough prepared from 1500 g of wheat flour, 400 Brabender Units (BU)of water, 90 g of yeast, 20 g of sucrose and 20 g of salt to which wasadded 0, 100, 250, 875 and 1250 units per kg of flour, respectively ofthe above hexose oxidase preparation. The measurement was carried outessentially in accordance with the calorimetric method of Ellman (1958)as also described in Cereal Chemistry, 1983, 70, 22-26. This method isbased on the principle that 5.5′-dithio-bis(2-nitrobenzoic acid) (DTNB)reacts with thiol groups in the dough to form a highly coloured anion of2-nitro-5-mercapto-benzoic acid, which is measuredspectrophotometrically at 412 nm.

Assuming that the relative change of the amount of thiol groups in adough is reflected as the change in the optical density (OD) resultingfrom the reaction between thiol groups and DTNB in the dough, thefollowing results were obtained:

Hexose oxidase GOD units/kg flour OD₄₁₂ 0 0.297 100 0.285 250 0.265 8750.187 1250 0.138

Thus, this experiment showed a significant decrease in OD indicating areduction of the content of free thiol groups which was proportionate tothe amount of hexose oxidase activity added.

2.4. Improvement of the Rheological Characteristics of Dough by theAddition of Hexose Oxidase

The above dough was subjected to extensigraph measurements according toAACC Method 54-10 with and without the addition of an amount of thehexose oxidase preparation corresponding to 100 units/kg flour of hexoseoxidase activity. The dough without addition of enzyme served as acontrol.

The principle of the above method is that the dough after forming issubjected to a load-extension test after resting at 30° C. for 45, 90,135 and 180 minutes, respectively, using an extensigraph capable ofrecording a load-extension curve (extensigram) which is an indication ofthe doughs resistance to physical deformation when stretched. From thiscurve, the resistance to extension, B (height of curve) and theextensibility, C (total length of curve) can be calculated. The B/Cratio (D) is an indication of the baking strength of the flour dough.

The results of the experiment is summarized in Table 2.1 below.

TABLE 2.1 Extensigraph measurements of dough supplemented with 100 GODunits/kg flour of hexose oxidase (HOX). Sample Time, min B C D = B/CControl 45 230 180 1.3 HOX 45 320 180 1.8 Control 90 290 161 1.8 HOX 90450 148 3.0 Control 135 290 167 1.7 HOX 135 490 146 3.4 Control 180 300168 1.8 HOX 180 500 154 3.2

It is apparent from this table that the addition of hexose oxidase (HOX)has an improving effect on the doughs resistance to extension asindicated by the increase in B-values. This is reflected in almost adoubling of the B/C ratio as a clear indication that the baking strengthof the flour is significantly enhanced by the hexose oxidase addition.

In a similar experiment, 100 units/kg flour of a commercial glucoseoxidase product was added and the above parameters measured in the samemanner using a dough without enzyme addition as a control. The resultsof this experiment is shown in Table 2.2 below:

TABLE 2.2 Extensigraph measurements of dough supplemented with 100 GODunits/kg flour of glucose oxidase (GOX). Sample Time, min B C D = B/CControl 45 240 180 1.3 GOX 45 290 170 1.7 Control 90 260 175 1.5 GOX 90360 156 2.3 Control 135 270 171 1.6 GOX 135 420 141 3.0

When the results for the above two experiments are compared with regardto differences between control dough and the hexose oxidase or glucoseoxidase supplemented doughs it appeared that hexose oxidase has astronger strengthening effect than glucose oxidase. Furthermore, the B/Cratio increased more rapidly with hexose oxidase relative to glucoseoxidase which is a clear indication that enhancement of the bakingstrength is being conferred more efficiently by hexose oxidase than byglucose oxidase (FIG. 1).

EXAMPLE 3

Dough Improving Effect of Hexose Oxidase Extracted from Chondrus crispus

For this experiment fresh Chondrus crispus seaweed fronds were harvestedalong the coast of Hirsholmene, Denmark. Hexose oxidase was isolatedusing two different extraction procedures, and the materials from bothwere pooled for the below dough improving experiment.

3.1 Purification of Hexose Oxidase from Chondrus crispus I

954 g of the fresh fronds was rinsed in distilled water, dried with atowel and stored in liquid nitrogen. The seaweed was blended using aWaring blender and 1908 ml of 0.1 M sodium phosphate buffer, 1 M NaCl,pH 6.8 was added to the blended seaweed. The mixture was extracted underconstant stirring for 4 days at 5° C., followed by centrifugation of themixture at 20,000×g for 30 minutes.

The resulting 1910 ml supernatant (351.1 U/ml) was concentrated to 440ml at 40° C. in a Büchi Rotavapor R110. The concentrate was ammoniumsulphate fractionated to 25%. The mixture was stirred for 30 minutes andcentrifuged for 20 minutes at 47,000×g. The supernatant (395 ml) wasdialysed overnight against 20 l of 10 mM triethanolamine (TEA) buffer,pH 7.3 to a final volume of 610 ml (367.1 U/ml).

The above 610 ml was applied in two runs to a 2.6×25 cm column with 130ml Q-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3. The columnwas washed with the equilibration buffer and the bound proteins wereeluted using 800 ml gradient from 0 to 0.8 M NaCl in equilibrationbuffer. The column was eluted at 4 ml/minute and fractions of 12 mlcollected. Fractions containing the hexose oxidase activity werecollected and pooled to a final volume of 545 ml (241.4 U/ml).

3.2. Purification of Hexose Oxidase from Chondrus crispus II

1250 g of the fresh fronds was rinsed in distilled water, dried with atowel and stored in liquid nitrogen. The seaweed was blended in a Waringblender followed by the addition of 2500 ml 0.1 M sodium phosphatebuffer, 1 M NaCl pH 6.8. The mixture was extracted under continuousstirring for 4 days at 50° C. followed by centrifugation at 20,000×g for30 minutes.

The resulting 2200 ml supernatant (332.S U/ml) was concentrated to 445ml 40° C. using a Büchi Rotavapor R110. The resulting concentrate wasammonium sulphate fractionated to 25%. The mixture was stirred for 30minutes and centrifuged for 20 minutes, at 47,000×g. The precipitate wasdiscarded. The 380 ml supernatant was dialysed overnight against 20 l 10mM TEA buffer, pH 7.3, to a final volume of 850 ml (319.2 U/ml).

The above 850 ml was applied to a 2.6×25 cm column with 130 mlQ-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3. The column waswashed with the equilibration buffer and the bound proteins were elutedusing 800 ml gradient from 0 to 0.8 M NaCl in equilibration buffer. Thecolumn was eluted at 4 ml/minute and fractions of 12 ml collected.Fractions containing the hexose oxidase activity were collected andpooled to a final volume of 288 ml.

The retentate from the above step was applied to a 2.6×31 cm column with185 ml metal chelating sepharose FF loaded with Ni²⁺ and equilibrated in50 mM sodium phosphate, 1 M NaCl, pH 7.4. The bound proteins were elutedwith a 740 ml gradient of 0 to 35 mM imidazole, pH 4.7 in equilibrationbuffer. The column was eluted at 2 ml/minute and fractions of 11 ml wascollected. Fractions 41-54 (140 ml, 352.3 U/ml) were pooled. Some hexoseoxidase did run through the column.

3.3. Pooling and Concentrating of Extracts

The run through and the 140 ml from purification II and the 545 ml frompurification I were pooled to a final volume of 1120 ml (303.6 U/ml).The 1120 ml was rotation evaporated into a volume of 210 ml followed bydialysis overnight against 20 l of 10 mM TEA buffer, pH 7.3, to a finalvolume of 207 ml (1200.4 U/ml).

3.3.1. Anion Exchange Step

The retentate resulting from the above step was applied to a 2.6×25 cmcolumn with 130 ml Q-sepharose -F equilibrated in 20 mM triethanolamine,pH 7.3. The column was washed with the equilibration buffer and thebound proteins eluted using 800 ml gradient from 0 to 0.8 M NaCl inequilibration buffer. The column was eluted at 4 ml/minute and fractionsof 12 ml collected. Fractions 30-50 containing the hexose oxidaseactivity (260 ml, 764.1 U/ml) were collected and pooled.

3.3.2. Other Enzyme Activity

The above pooled solution was tested for the following enzymatic sideactivities catalase, protease, xylanase, α and β-amylase and lipase.None of these activities were found in the solution.

3.4. Improvement of the Rheological Characteristics of Dough by theAddition of Hexose Oxidase

A dough was prepared from wheat flour, water and salt and 0, 72, 216 and360 units per kg of flour, respectively of the above hexose oxidasepreparation was added hereto. The dough without addition of enzymeserved as a control. In addition two doughs were prepared to which wasadded 216 and 360 units per kg of flour respectively, of Gluzyme, aglucose oxidase available from Novo Nordisk A/S, Denmark.

The doughs were subjected to extensigraph measurements according to amodification of the above ACC Method 54-10. The results of theexperiment are summarized in Table 3.1 below.

TABLE 3.1 Extensigraph measurements of dough supplemented with hexoseoxidase (HOX) or glucose oxidase (units per kg flour) Sample Time, min.B C D = B/C Control 45 250 158 1.6 HOX 72 U/kg 45 330 156 2.1 HOX 216U/kg 45 460 153 3.0 HOX 360 U/kg 45 580 130 4.5 Gluzyme 72 U/kg 45 350159 2.2 Gluzyme 216 U/kg 45 340 148 2.3 Gluzyme 360 U/kg 45 480 157 3.1Control 90 290 164 1.8 HOX 72 U/kg 90 470 145 3.2 HOX 216 U/kg 90 650142 4.6 HOX 360 U/kg 90 870 116 7.5 Gluzyme 72 U/kg 90 450 147 3.1Gluzyme 216 U/kg 90 480 138 3.5 Gluzyme 360 U/kg 90 500 152 3.2 Control135 330 156 2.1 HOX 72 U/kg 135 540 129 4.2 HOX 216 U/kg 135 750 125 6.0HOX 360 U/kg 135 880 117 7.5 Gluzyme 72 U/kg 135 510 136 3.8 Gluzyme 216U/kg 135 550 122 4.5 Gluzyme 360 U/kg 135 560 121 4.6

It is evident from the above table that the addition of hexose oxidase(HOX) or glucose oxidase had an improving effect on The resistance ofdoughs to extension as indicated by the increase in B-values. This isreflected in an increase of the B/C ratio as a clear indication that thebaking strength of the flour was enhanced significantly by the additionof enzymes.

It is also evident that the hexose oxidase had a higher strengtheningeffect than glucose oxidase. Furthermore, the B/C ratio increased morerapidly with hexose oxidase relative to glucose oxidase which is a clearindication that enhancement of the baking strength is being conferredmore efficiently by hexose oxidase than by glucose oxidase.

EXAMPLE 4

Dough Improving Effect of Hexose Oxidase Extracted from Chondrus crispus

4.1. Purification of Hexose Oxidase from Chondrus crispus

Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. 2285 g of this fresh material was rinsed in distilledwater, dried with a towel and stored in liquid nitrogen. The seaweed wasblended in a Waring blender followed by addition of 4570 ml 0.1 M sodiumphosphate buffer, 1 M NaCl pH 6.8. The mixture was extracted undercontinuous magnetic stirring for 4 days at 5° C. followed bycentrifugation at 20,000×g for 30 minutes.

The resulting 4930 ml supernatant (624.4 U/ml) was concentrated to 1508ml at 40° C. using a Büchi Rotavapor R110. The obtained concentrate waspolyethylenglycol fractionated to 3% (w/v). The mixture was stirred for30 minutes and centrifuged for 30 minutes at 47,000×g. The pellet wasdiscarded. The 1470 ml supernatant (2118.7 U/ml) was PEG fractionated to24%. The mixture was stirred for 30 minutes and centrifuged for 30minutes at 47,000×g. The supernatant was discarded and the 414.15 g ofprecipitate was resuspended in 200 ml 20 mM TEA buffer, pH 7.3, followedby dialysis over night at 5° C. against 20 l 10 mM TEA buffer, pH 7.3.

After dialysis the volume was 650 ml (2968.6) U/ml). The suspension wascentrifuged for 30 minutes at 20,000×g. The precipitate was discardedand the supernatant was diluted to 3200 ml with distilled water.

The above 3200 ml (829.9 U/ml) was applied to a 10×14 cm column with1100 ml Q-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3. Thecolumn was washed with the equilibration buffer and the bound proteinswere eluted using 15,000 ml gradient from 0 to 0.8 M NaCl inequilibration buffer. The column was eluted at 50 ml/minute. Hexoseoxidase did run through the column and 840 ml of this was collected.

The 840 ml suspension was treated with kieselguhr and concentrated to335 ml (2693.3 U/ml).

The above 335 ml was applied to a 3 l Sephadex G25C desalting column10×40 cm. The column was equilibrated in 20 mM TEA buffer, pH 7.3,eluted at a flow rate of 100 ml/minute and 970 ml eluate was collected.This eluate was applied to a 10×14 cm column with 1100 ml Q-Sepharose FFequilibrated in 20 mM TEA, pH 7.3. The column was washed with theequilibration buffer and bound proteins eluted using a 15,000 mlgradient of 0 to 0.8 M NaCl in equilibration buffer. The column waseluted at 50 ml/min. Hexose oxidase did run through the column and 1035ml of this was collected.

To the above eluate (1035 ml) ammonium sulphate was added to a finalconcentration of 2 M. The mixture was then applied in two runs to a 5×10cm column with 200 ml phenyl sepharose HP equilibrated in 25 mM sodiumphosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄. The column was washed withequilibration buffer followed by eluting the bound proteins at a flowrace of 50 ml/minute using 5,000 ml gradient from 2 M to 0 M NH₄)₂SO₄ in25 mM sodium phosphate buffer. Fractions of 500 and 29 ml, respectivelywere collected from run 1 and 2. Fraction 5 in run 1 and fractions 27-42in run 2 containing the hexose activity were pooled to a total of 1050ml (563.9 U/ml).

The above pool was desalted by a 3 l Sephadex G25C gel filtrationcolumn. The column was equilibrated in 20 mM TEA buffer, pH 7.3, elutedat a flow rate of 100 ml/minute and 1,000 ml eluate was collected.

The 1,000 ml eluate was concentrated to 202 ml (2310.2 U/ml) and thispreparation was used for following rheology testing.

4.2. Improvement of the Rheological Characteristics of Dough by theAddition of Hexose Oxidase

A dough was prepared from wheat flour, water and salt and 0, 288, 504and 720 oxidoreductase units per kg of flour, respectively of the abovehexose oxidase preparation was added hereto. The dough without additionof enzyme served as a control. In addition two doughs were prepared towhich was added 288 and 504oxidoreductase units per kg of flourrespectively, of Gluzyme, a glucose oxidase available from Novo NordiskA/S, Denmark.

The doughs were subjected to extensigraph measurements according to amodification of AACC Method 54-10.

The results of the experiment are summarized in Table 4.1 below.

TABLE 4.1 Extensigraph measurements of dough supplemented with hexoseoxidase (HOX) or glucose oxidase (Units per kg flour). Sample Time, min.B C D = B/C Control 45 210 171 1.2 HOX 288 U/kg 45 490 139 3.5 HOX 504U/kg 45 640 122 5.2 HOX 720 U/kg 45 730 109 6.7 Gluzyme 288 U/kg 45 350165 2.1 Gluzyme 504 U/kg 45 385 153 2.5 Gluzyme 720 U/kg 45 435 148 2.9Control 90 275 182 1.5 HOX 288 U/kg 90 710 130 5.5 HOX 504 U/kg 90 825106 7.8 HOX 720 U/kg 90 905 107 8.5 Gluzyme 288 U/kg 90 465 153 3.0Gluzyme 504 U/kg 90 515 135 3.8 Gluzyme 720 U/kg 90 540 140 3.9 Control135 280 175 1.6 HOX 288 U/kg 135 745 102 7.3 HOX 504 U/kg 135 920 94 9.8HOX 720 U/kg 135 — 80 — Gluzyme 288 U/kg 135 525 129 4.1 Gluzyme 504U/kg 135 595 129 4.6 Gluzyme 720 U/kg 135 630 121 5.2

It is apparent from the above results that the addition of hexoseoxidase (HOX) or glucose oxidase has an improving effect on theresistance of doughs to extension as indicated by the increase inB-values. This is reflected in an increase of the B/C ratio.

It is also apparent that hexose oxidase has a stronger strengtheningeffect than that of glucose oxidase, the strengthening effect of bothenzymes being proportional to the amount of enzyme added. Furthermore,the B/C ratio increased more rapidly with hexose oxidase relative toglucose oxidase which is a clear indication that enhancement of thebaking strength is being conferred more efficiently by hexose oxidasethan by glucose oxidase.

EXAMPLE 5

Improving Effect of Hexose Oxidase Extracted from Chondrus crispus onthe Specific Volume of Bread

5.1. Purification of Hexose Oxidase from Chondrus crispus

Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. 2191 g of this fresh material was rinsed in distilledwater, dried with a towel and stored in liquid nitrogen. The seaweed wasblended in a Waring blender followed by addition of 4382 ml 0.1 M sodiumphosphate buffer, 1 M NaCl and pH 6.8. The mixture was extracted undercontinuously magnetic stirring for 4 days at 5° C. followed bycentrifugation at 20,000×g for 20 minutes.

The resulting 4600 ml supernatant (746.1 U/ml) was concentrated to 850ml at 40° C. in a Büchi Rotavapor R110. This concentrate (3626.9 U/ml)was polyethylene glycol fractionated to 3% (w/v). The mixture wasstirred for 30 minutes and centrifuged for 30 minutes at 20,000×g. Theprecipitate was discarded. The. 705 ml supernatant (2489.8 U/ml) was PEGfractionated to 25%. The mixture was stirred for 30 minutes andcentrifuged for 30 minutes at 20,000×g. The supernatant was discardedand the 341 g of precipitate was resuspended in 225 ml 20 mM TEA buffer,pH 7.3. The suspension (500 ml) was desalted on a 3 l Sephadex G25Cdesalting column 10×40 cm. The column was equilibrated in 20 mM TEAbuffer, pH 7.3, and eluted at a flow rate of 100 ml/minute. 1605 mleluate was collected.

To the above eluate (687.5 U/ml) ammonium sulphate was added to a finalconcentration of 2 M. The mixture was then applied in two runs to a 5×10cm column with 200 ml phenyl sepharose HP equilibrated in 25 mM sodiumphosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄. The column was washed withequilibration buffer followed by elution of the bound proteins at a flowrate of 50 ml/minute using 5,000 ml gradient from 2 M to 0 M (NH₄)₂SO₄in 25 mM sodium phosphate buffer. Fractions of 29 ml was collected.Fractions 85-105 in run 1 and fractions 36-69 in run 2 containing thehexose activity were pooled to a total of 1485 ml (194.7 U/ml)

The above pool was desalted by a 3 l Sephadex G25C gelfiltration column,the same as used in 4.1. The column was equilibrated in 20 mM TEAbuffer, pH 7.3, and eluted at a flow rate of 100 ml/minute. 1,200 mleluate was collected.

The 1,200 ml eluate was concentrated to 685 ml (726.2 U/ml) and used forbaking experiments.

5.2. Improvement of the Specific Volume of Bread by Adding HexoseOxidase to the Dough

A dough was prepared from 1500 g of flour, 90 g of yeast, 24 g of salt,24 g of sugar and 400 BU of water and 0 or 108 units of the abovepurified hexose oxidase and 108 units of Gluzyme (glucose oxidaseavailable from Novo Nordisk, Denmark) per kg flour, respectively wasadded hereto. The dough was mixed on a Hobart mixer for 2+9 minutes at26° C. and divided into two parts followed by resting for 10 minutes at30° C. in a heating cabinet, moulding with a Fortuna 3/17/7 and proofingfor 45 minutes at 34° C. and 85% RH. The thus proofed dough was baked at220° C. for 17 minutes with 12 sec. steam in a Bago oven.

The results of the experiment are summarized in table 5.1 below.

TABLE 5.1 Improvement of specific volumes of bread prepared from doughsupplemented with hexose oxidase or glucose oxidase (Units per kg flour)Total Total Specific volume weight volume Control 5325 1027 5.18 Hexoseoxidase 108 U/kg 6650 1036 6.41 Gluzyme 108 U/kg 6075 1030 5.89

It is evident from the above table that the addition of hexose oxidaseor glucose oxidase had an increasing effect on the total volume, theweight being essentially the same. This is reflected in an increase ofthe specific volume as compared to the bread baked without addition ofenzymes.

It is also evident that hexose oxidase has a significantly larger effecton the increase of the specific volume than had glucose oxidase at thesame dosage.

EXAMPLE 6

Characterization of the Purified Hexose Oxidase

Preparations from the above purifications were used for characterizationof hexose oxidase.

6.1. Staining for Hexose Activity After Non-Denaturing PAGE

Hexose oxidase activity was analyzed by native PAGE using precast 8-16%Tris-glycine Novex gels according to the manufactures instructions(Novex, San Diego, USA). After electrophoresis the gels were stained forhexose activity by incubation of the gel in a solution containing 50 mMsodium phosphate buffer, pH 6.0, 100 mM glucose, 50 mg/l phenazinemethosulphate (Sigma, P9625) and 250 mg/l nitroblue tetrazolium (SigmaN6876) as described in the PhD thesis by Witteveen, C. F. B. (1993)“Gluconate formation and polyol metabolism in Aspergillus niger”. Afterabout 30 minutes the hexose activity was visible as a double bond veryclose to each other. The same double band was also seen when a nativePAGE of hexose oxidase was silver stained. The molecular weight ofpurified hexose oxidase was determined to 144 kD by native PAGE. Halfthe gel was silver stained, the other half was activity stained. Asstandards were used bovine serum albumin (67 kD), lactate dehydrogenase(140 kD), catalase (232 kD), ferritin (440 kD) and thyroglobulin (669kD).

6.2 Determination of Molecular Weight by SDS-Page

The molecular weight was also determined on material which was firstapplied to a native PAGE as described above, after activity staining thehexose oxidase band was excised from the gel and then electroelutedusing an Electro-Eluter (model 422, Bio-Rad, CA, USA) according to themanufacturer's recommendations. The electroeluted protein was subjectedto SDS-PAGE and silver stained. This material gave “one” double bond atabout 70 kDa in SDS-PAGE gels. The electroeluted hexose oxidase istherefore a dimer of two subunits.

6.3 Determination of pI of Hexose Oxidase

Samples containing hexose oxidase activity were analyzed by isoelectricfocusing (IEF) using a precast 3-10 IEF gel according to themanufacturer's recommendations (Novex, San Diego, US). Afterelectrophoresis half of the gel was silver stained and the other halfnitroblue tezrazolium stained as described in 6.1.

Hexose oxidase stained as a double band. The pI of the first band was4.79, pI of the second band was 4.64. As standards were used trypsinogen(9.30), lentil lectin basic band (8.65), lentil lectin middle band(8.45), lentil lectin acid band (8.15), horse myoglobin acidic band(6.85), human carbonic anhydrase B (5.85), β-lactoglobulin A (5.20), soybean trypsin inhibitor (4.55) and amyloglucosidase (3.50).

6.4 Determination of K_(m) of Hexose Oxidase for Different Sugars

K_(m) of hexose oxidase was determined for 7 different sugars asdescribed in 1.2.3. Results are summarized in table 6.1 below.

TABLE 6.1 Determination of K_(m) of hexose oxidase for different sugarsSubstrate K_(m) (mM) CV (mM) D-glucose 2.7 0.7 D-galactose 3.6 1cellobiose 20.2 7.8 maltose 43.7 5.6 lactose 90.3 20.6 xylose 102 26arabinose 531 158 (CV = coefficient of variation)6.5 Determination of a Peptide Sequence of the Hexose Oxidase

50 μl from the electroeluted mixture in 6.2 was suspended in 450 μl 0.1%triflouracetic acid (TFA).

To remove the Tris, glycine and SDS, the above mixture was subjected tochromatography on reverse-phase HPLC. The resulting solution was appliedin 9 runs to a 4.6×30 cm Brownlee C2 column equilibrated in 0.1% TFA.The column was washed in equilibration buffer and bound peptides elutedwith a 14 ml gradient from 10 to 80% acetonitrile in 0.1% TFA, at a flowrate of 0.7 ml/min. Fractions from the largest peak containing theenzyme were collected and freeze dried.

6.5.1 Endoproteinase Lys-C Digestion

Resulting freeze dried enzyme was dissolved in 50 μl 8 M urea, 0.4 MNH₄HCO₃, pH 8.4. Denaturation and reduction of the protein was carriedout by the addition of 5 μl mM dithiothreitol and under an overlay of N₂at 50° C. for 15 min. The solution was cooled to room temperature and 5μl 100 mM iodoacetamide was added, the cysteines being derivatized for15 min. at room temperature in the dark under N₂. Subsequently, thesolution was suspended in 135 μl water and digestion was carried out at37° C. under N₂ for 24 hours by addition of 5 μg endoproteinase Lys-Cdissolved in 5 μl water. The reaction was terminated by freezing thereaction mixture at −20° C.

6.5.2 Reverse-Phase HPLC Separation of Peptides

The resulting peptides were separated by reverse-phase HPLC on a VYDACC18 column 0.46×15 cm (The Separation Group, CA, USA) using as solvent A0.1% TFA in water and as solvent B 0.1% TFA in acetonitrile.

6.5.3 Peptide Sequencing

Sequencing was performed on an Applied Biosystems 476A sequencer(Applied Biosystems, CA, USA) using pulsed-liquid fast cycles accordingto the manufacturer's instructions. A peptide having the below aminoacid sequence was identified:

D P G Y I V I D V N A G T P D K P D P.

1. A dough improving composition comprising an oxidoreductase which isat least capable of oxidizing maltose, flour and at least one furtherdough ingredient or dough additive wherein said oxidoreductase is in anamount which results in the presence in a finished dough of 1 to 10,000units per kg of flour.
 2. A composition according to claim 1 wherein theoxidoreductase is derived from a source selected from an algal species,a plant species and a microbial species.
 3. A composition according toclaim 2 wherein the oxidoreductase is hexose oxidase.
 4. A compositionaccording to claim 3 wherein the hexose oxidase is derived from Chondruscrispus.
 5. A composition according to claim 1 which is a pre-mixtureuseful for preparing a baked product or in making a noodle product or analimentary paste product.
 6. A composition according to claim 1 whichcomprises an additive selected from the group consisting of anemulsifying agent and a hydrocolloid.
 7. A composition according toclaim 6 wherein the hydrocolloid is selected from the group consistingof an alginate, a carrageenan, a pectin and a vegetable gum.
 8. A doughcomprising a dough improving composition comprising an oxidoreductasewhich is at least capable of oxidizing maltose and at least one furtherdough ingredient or dough additive, and flour, wherein saidoxidoreductase is in an amount which results in the presence in thefinished dough of 1 to 10,000 units per kg of flour.
 9. A flour doughcomprising an oxidoreductase which is at least capable of oxidizingmaltose and flour, wherein said oxidoreductase is in an amount whichresults in the presence in the finished dough of 1 to 10,000 units perkg of flour.
 10. The flour dough according to claim 9 wherein said flouris selected from the group consisting of wheat flour, rice flour, maizeflour, barley flour, rye flour, durra flour and mixtures thereof. 11.The flour dough according to claim 9 wherein said flour dough comprisesat least one further enzyme.
 12. The flour dough according to claim 9wherein said flour dough comprises at least one further enzyme andwherein said further enzyme is selected from the group consisting ofcellulase, a hemicellulase, a xylanase, a starch degrading enzyme, aglucose oxidase, a lipase and a protease.
 13. The flour dough accordingto claim 9 wherein said oxidoreductase is hexose oxidase.
 14. The flourdough according to claim 9 wherein said oxidoreductase is hexose oxidaseand wherein said hexose oxidase is derived from a source selected fromthe group consisting of an algal species, a plant species and amicrobial species.
 15. The flour dough according to claim 9 wherein saidoxidoreductase is hexose oxidase and wherein said hexose oxidase isderived from Chondrus crispus.
 16. A baked or dried product producedfrom a flour dough wherein said flour dough comprises an oxidoreductasewhich is at least capable of oxidizing maltose, wherein saidoxidoreductase is in an amount which results in the presence in thefinished dough of 1 to 10,000 units per kg of flour.
 17. The baked ordried product according to claim 16 wherein said flour is selected fromthe group consisting of wheat flour, rice flour, maize flour, barleyflour, rye flour, durra flour and mixtures thereof.
 18. The baked ordried product according to claim 16 wherein said flour dough comprisesat least one further enzyme.
 19. The baked or dried product according toclaim 16 wherein said flour dough comprises at least one further enzymeand wherein said further enzyme is selected from the group consisting ofcellulase, a hemicellulase, a xylanase, a starch degrading enzyme, aglucose oxidase, a lipase and a protease.
 20. The baked or dried productaccording to claim 16 wherein said oxidoreductase is hexose oxidase. 21.The baked or dried product according to claim 16 wherein saidoxidoreductase is hexose oxidase and wherein said hexose oxidase isderived from a source selected from the group consisting of an algalspecies, a plant species and a microbial species.
 22. The baked or driedproduct according to claim 16 wherein said oxidoreductase is hexoseoxidase and wherein said hexose oxidase is derived from Chondruscrispus.
 23. The baked product according to claim 16 wherein said bakedproduct is a bread product.
 24. The dried product according to claim 16wherein said dried product is a noodle or an alimentary paste product.